Autosomal dominant polycystic kidney disease (ADPKD), which is characterized by marked phenotypic variability, is caused by mutation to PKD1 or PKD2. Understanding factors that underlie this variability is of diagnostic, prognostic and mechanistic importance. During the past funding period we have improved mutation detection levels from ~70-90% by employing direct sequencing, evaluation of non-definite variants for pathogenicity, and screening for larger rearrangements. The remaining ~10% of cases will be a major focus of Aim 1 of this proposal to understand the etiology of disease. Areas that will be explored are: atypical mutations to regulatory regions, including potential miRNA binding sites; cryptic splicing; mosaicism; gene conversion to PKD1; and further genetic heterogeneity. A group of ~170 families without detected conventional PKD1 or PKD2 mutations will be available, as well as access to putative unlinked families. A major finding from the previous study period was that incompletely penetrant (hypomorphic) PKD1 alleles underlie some of the phenotypic variability in ADPKD. Alone they can be responsible for mild PKD1, while as homozygotes or compound heterozygotes (including in trans with an inactivating allele), can result in early-onset disease. The full role of these incompletely penetrant alleles will be analyzed here be determining their frequency in populations with either later or early-onset ADPKD (Aim 2). How these alleles exert an effect will be analyzed at the cellular level. To fully prove their significance and test the mode of action, knock-in mouse models of two variants will be generated. The role of the alleles in modulating the phenotype of the whole animal will be tested and by combining alleles (including with existing null alleles) models with different disease severities will be generated; suitable for therapeutic testing. Expression of the hypomorphic alleles at the mRNA and protein level will be assayed, and the cellular consequences assessed. The last aim will explore next-generation sequencing methods to improve mutation screening of ADPKD. Protocols will be developed to further analyze the mutation negative patient group, specifically looking for changes missed by the existing locus-specific analysis of PKD1 and conventional sequencing. Finally, a more comprehensive screening protocol including related disease genes and potential modifier loci will be developed for ADPKD diagnostic/prognostic screening. The proposed studies should, overall, reveal more about mechanisms of mutation causing ADPKD, including the significance of further genetic heterogeneity and hypomorphic alleles, and result in a much more comprehensive screening protocol.